Presentation on theme: "Therapeutic use of hair follicle-derived epithelial stem cells using a murine stem cell deficiency model Slide 1 The authors have no financial interest."— Presentation transcript:
1Therapeutic use of hair follicle-derived epithelial stem cells using a murine stem cell deficiency modelSlide 1The authors have no financial interest in the subject matter of this posterEwa Anna Meyer-Blazejewska, Hongshan Liu, Mindy K. Call,Ursula Schlötzer-Schrehardt,Winston W-Y. Kao and Friedrich E. Kruse1 Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany2 Department of Ophthalmology, University of Cincinnati, OH, USA
2Introduction: Hair follicle stem cells Slide 2Introduction: Hair follicle stem cellsMurine HFThe bulge region of the hair follicle (HF) is a major reservoir of multipotent adult stem cells (SC). (Cotsarelis et al. 1990)Cytokeratin 15 (K15), a marker for stem and progenitor cells in the bulge and outer root sheath of the hair follicle.(Cotsarelis et al., 1990, 1999; Fiqueira et al., 2007)bulge
3Introduction: Hair follicle markers Slide 3Introduction: Hair follicle markersNo expression of the corneal epithelial markers(K12, Pax6) in hair follicleHair follicleK12-Pax6-Sebaceous glandinner root sheathCorneabulgeK12+outerroot sheathPax6+/K12+
4Introduction: previous work Slide 4Induction of K12 and Pax6 expression in hair follicle SC in vitro using conditioned medium (CM) derived from limbal stroma fibroblastsn=5n=5Cytokeratin 12Pax 6********molecules K12/molecules ß-actin x103molecules Pax6/molecules ß-actin x103centralcornealfibroblastCMperipheralcornealfibroblastCMlimbalfibroblastCM3t3fibroblastCMcentralcornealfibroblastCMperipheralcornealfibroblastCMlimbalfibroblastCM3t3fibroblastCMK12K12/Pax6Blazejewska et al.; Stem Cells, 2008
5Purpose To explore the therapeutic potential Slide 5PurposeTo explore the therapeutic potentialof murine hair follicle-derived stem cellsto treat limbal stem cell deficiency and replenish corneal epitheliumusing an in vivo animal model.
6Inducible K12 driven expression of EGFP Slide 6Method:Tri-Transgenic Mouse ModelInducible K12 driven expression of EGFPcrertTAK12IRESrtTApminCMVcretet-ODoxDoxDoxDoxrtTApCAmTmGlox Plox PpCAmG
7Method:Tri-Transgenic Mouse Model Slide 7Method:Tri-Transgenic Mouse ModelWe have generated a tri-transgenic mouse model that is both tissue specific and inducible and allows for the detection of K12 expressing cells by the presence of green fluorescence. This transgenic mouse system is comprised of three parts the first of which is the K12 rtTA line that provides the tissue specificity. This line was generated via a knock-in strategy in which an IRES-rtTA (Internal Ribosome Entry Site-reverse tetracycline Transcriptional Activator) minigene was inserted directly after the stop codon of the mouse Krt12 gene. Thereby only differentiated corneal epithelial cells are able to express rtTA. The second component of the tri-transgenic mouse model is Tet-O-Cre. This line uses components of the Tet-On system and together with the K12 rtTA line provides the ability for induction. Specific Tetracycline operator (Tet-O) elements are followed by a CMVmin (CMV minimal) promotor and the Cre recombinase gene. In the absence of tetracycline or a tetracycline derivate such as doxycycline , rtTA is unable to bind to the promotor and therefore Cre is not produced. Once doxycycline is added to the system, it can bind with rtTA and together this complex can further bind to the Tet-O elements and drive the expression of Cre. The third component of the system is the ROSA26mTmG line (Jackson Laboratories) which serves as a dual reporter. This mouse line has loxP sites flanking a membrane-targeted tdTomato (mT) cassette and express red fluorescence in all cell types. Upon breeding to a Cre recombinase mouse line (Tet-O-Cre), the resulting offspring will have td Tomato cassette deleted only in the cells expressing Cre (only in K12 positive cells) allowing for expression of a membrane-targeted enhanced green fluorescent protein (mG). This system allows for the live visualization and tracking of K12 expressing cells.
8Method: Clonal expansion of hair follicle SC Slide 8Method: Clonal expansion of hair follicle SCStem cell clones grown on a 3T3 feeder layerSC cloneSC clones3T3 cellsSC cloneK15EpithelialcellsRed fluorescence: no K12 expression in HF-derived epithelial SC clonesSC cloneK12rtTA/Tet-O-Cre/ROSAmTmGZ-stack, 3D
9Method: Transplantation of SC on a fibrin gel Slide 9Method: Transplantation of SC on a fibrin gelAfter limbal SC debridementSC clones subcultured on a fibrin gel as carrierK12rtTA/Tet-O-Cre/ROSAmTmGDirectly after SC transplantationRed:SC and progenitor cellsNo Green:no K12 expressionFibrin gel
10Results: K12 induction post-transplantation Slide 10Results: K12 induction post-transplantation3 days postoperativeMouse eye21 days postoperativeFibrin gelremains14 days postoperativeRegular lightFluorescein stainingK12+ cellsWT C57/Black6
11Results: K12 induction post-transplantation Slide 11Corneal epithelium: 7days postoperativeDAPIEGFPK12+ (green)tdTomatoMergeno K12 (red)The specimen was prepared by removing the cornea, treating with 0.2% sodium borohydride for 45 min at room temperature (helps in the reduction of background fluorescence), counterstaining with DAPI overnight, and imaging. The total thickness of the Z-stack is 37.5 µm with each slice having a thickness of 1.5 µm. All images are from slice 14 of the Z-stack.
12Slide 12ConclusionsHair follicle bulge-derived epithelial SC possess the potential to differentiate into corneal epithelial-like phenotype in vivo.Hair follicle SC express K12 (corneal epithelial differentiation marker) and regenerate the corneal epithelium up to 3 weeks post-transplantation when transplanted in a murine limbal SC deficiency model.